J Clin Med Res

Journal of Clinical Medicine Research, ISSN 1918-3003 print, 1918-3011 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Med Res and Elmer Press Inc

Journal website http://www.jocmr.org

Original Article

Volume 11, Number 2, February 2019, pages 98-105

Cytoskeletal Tropomyosin as a Biomarker in Clostridium difficile Infection

Figure 2. Distribution of samples tested for tropomyosin. CDI: C. difficile infection; ID: an infectious disease physician; OEP: other enteric pathogens; “Tpm^-”: tropomyosin is not detected; “Tpm⁺”: tropomyosin is detected; “CDI⁺”: specimens tested positive by rtPCR (the Xpert^® C. difficile/Epi, Cepheid); “CDI^-”: specimens tested negative by rtPCR (the Xpert^® C. difficile/Epi, Cepheid); “true CDI”: compatible with CDI according to the reference approach (microbiology and clinical evaluation); “not CDI”: deviated from the reference approach; “Uncertain”: specimens tested positive by PCR but the diagnosis is inconclusive; “Consented”: sample was collected from consented patient. In the groups of “CDI^-” and “OEP”, clinical signs were evaluated only for “Tpm⁺” samples.

Figure 3. Detection of non-muscle tropomyison (Tpm) in stool samples. Samples #8-11 were collected from CDI patients. Samples #9-11 are positive for Tpm (upper bend). #11a: after the treatment, upper band is disappeared. The mixture of anti-Tpm antibodies (Abs) is comprised of monoclonal antibodies for Tpm1, Tpm 5, and chicken leg muscle Tpm. To control a loading amount of proteins an equal amount of stool starting material was always used for Tpm crude extraction.

**Table 1.** Tpm Detection and Distribution Within “CDI⁺” Group
	True CDI, n	Not CDI, n	Uncertain diagnosis, n	Total
In brackets % of total samples in corresponding categories from total samples in “CDI⁺” group. Underlined numbers are the most important indications. “true CDI” - “CDI⁺” subgroup: it denotes specimens collected from patients clinically evaluated as having real infection. “not CDI” - “CDI⁺” subgroup: it denotes samples collected from patients tested positive by PCR but CDI diagnosis was not confirmed. “Uncertain diagnosis”: specimens tested positive by PCR but the diagnosis is inconclusive.
Tpm positive	36	3	6	45 (24%)
Tpm negative	31	40	72	143 (76%)
Total	67 (35.6%)	43 (23%)	78 (41.5%)	188

Table 1. Tpm Detection and Distribution Within “CDI⁺” Group

True CDI, n

Not CDI, n

Uncertain diagnosis, n

Total

In brackets % of total samples in corresponding categories from total samples in “CDI⁺” group. Underlined numbers are the most important indications. “true CDI” - “CDI⁺” subgroup: it denotes specimens collected from patients clinically evaluated as having real infection. “not CDI” - “CDI⁺” subgroup: it denotes samples collected from patients tested positive by PCR but CDI diagnosis was not confirmed. “Uncertain diagnosis”: specimens tested positive by PCR but the diagnosis is inconclusive.

Tpm positive

45 (24%)

Tpm negative

143 (76%)

Total

67 (35.6%)

43 (23%)

78 (41.5%)

188

**Table 2.** Tpm Detection and Distribution Within “CDI⁺” Group in Inpatients
	True CDI, n	Not CDI, n	Uncertain diagnosis, n	Total
“true CDI” - “CDI⁺” subgroup: it denotes specimens collected from inpatients clinically evaluated as having real infection. “not CDI” - “CDI⁺” subgroup: it denotes samples collected from inpatients tested positive by PCR but CDI diagnosis was not confirmed. “Uncertain diagnosis”: specimens tested positive by PCR, but diagnosis is inconclusive.
Tpm positive	9	1	1	11
Tpm negative	9	13	16	38
Total	18	14	17	49

Table 2. Tpm Detection and Distribution Within “CDI⁺” Group in Inpatients

True CDI, n

Not CDI, n

Uncertain diagnosis, n

Total

“true CDI” - “CDI⁺” subgroup: it denotes specimens collected from inpatients clinically evaluated as having real infection. “not CDI” - “CDI⁺” subgroup: it denotes samples collected from inpatients tested positive by PCR but CDI diagnosis was not confirmed. “Uncertain diagnosis”: specimens tested positive by PCR, but diagnosis is inconclusive.

Tpm positive

Tpm negative

Total

**Table 3.** Clinical Stool Specimens Tested for Tropomyosin (Tpm)
	Tested samples, n	Tpm positive, n (%, in corresponding category)	Samples with available clinical signs, n
: only Tpm-positive samples; CDI⁺: remnant diarrheal stool specimens submitted to clinical microbiology laboratory that tested positive for C. difficile* toxin DNA by PCR method (Xpert^® C. difficile/Epi); CDI^-: emnant diarrheal stool specimens submitted to clinical microbiology laboratory that tested negative for C. difficile toxin DNA by PCR method (Xpert^® C. difficile/Epi); OEP: specimens tested for the presence of other enteric pathogens or parasites by routine testing methods.
CDI^-	228	22 (9.7)	19*
CDI⁺	230	56 (24.3)	188
OEP	52	12 (23)	10*
Total	510	90 (17.7)	217

Table 3. Clinical Stool Specimens Tested for Tropomyosin (Tpm)

Tested samples, n

Tpm positive, n (%, in corresponding category)

Samples with available clinical signs, n

*: only Tpm-positive samples; CDI⁺: remnant diarrheal stool specimens submitted to clinical microbiology laboratory that tested positive for C. difficile toxin DNA by PCR method (Xpert^® C. difficile/Epi); CDI^-: emnant diarrheal stool specimens submitted to clinical microbiology laboratory that tested negative for C. difficile toxin DNA by PCR method (Xpert^® C. difficile/Epi); OEP: specimens tested for the presence of other enteric pathogens or parasites by routine testing methods.

CDI^-

228

22 (9.7)

19*

CDI⁺

230

56 (24.3)

188

OEP

12 (23)

10*

Total

510

90 (17.7)

217

**Table 4.** Summary of Fecal Inflammatory Biomarkers as Possible Predictors of CDI Disease*
Biomarker	Clinical indication/prediction	Role in immunopathogenesis	Specific/sensitive to CDI
*: adapted from reference [10]. Permission was granted. pMK2: pyruvate kinase M2; IL-8: interleukin-8.
Lactoferrin	Colonic inflammation, CDI severity (when the level is elevated)	The innate inflammatory response; related to level of neutrophils translocation	no/no
Calprotectin	Intestinal inflammatory conditions (when the level is elevated); CDI severity	The innate inflammatory response: correlates with level of released neutrophils	no/no
IL-8	CDI severity (when elevated)	Involved in the recruitment of neutrophils to sites of infection	no/yes
IL-23	May relate to CDI recurrence (when the level is decreased)	The lack of a robust immunological response	no/no
pMK2	The presence of toxigenic C. difficile (when the level is elevated)	A key mediator of p38-dependent inflammation	no/-
pp38	Symptomatic CDI in pediatrics (when the level is elevated)	Activation of p38 protein pathway	yes/no

Table 4. Summary of Fecal Inflammatory Biomarkers as Possible Predictors of CDI Disease*

Biomarker

Clinical indication/prediction

Role in immunopathogenesis

Specific/sensitive to CDI

*: adapted from reference [10]. Permission was granted. pMK2: pyruvate kinase M2; IL-8: interleukin-8.

Lactoferrin

Colonic inflammation, CDI severity (when the level is elevated)

The innate inflammatory response; related to level of neutrophils translocation

no/no

Calprotectin

Intestinal inflammatory conditions (when the level is elevated); CDI severity

The innate inflammatory response: correlates with level of released neutrophils

no/no

IL-8

CDI severity (when elevated)

Involved in the recruitment of neutrophils to sites of infection

no/yes

IL-23

May relate to CDI recurrence (when the level is decreased)

The lack of a robust immunological response

no/no

pMK2

The presence of toxigenic C. difficile (when the level is elevated)

A key mediator of p38-dependent inflammation

no/-

pp38

Symptomatic CDI in pediatrics (when the level is elevated)

Activation of p38 protein pathway

yes/no